Polymorphisms in cg2 and pfcrt genes and resistance to chloroquine and other antimalarials in vitro in Plasmodium falciparum isolates from Colombia

Polymorphisms in Plasmodium falciparum cg2 and pfcrt genes and their association with chloroquine resistance in vitro in Colombian parasites were evaluated in this study. Association of chloroquine resistance with resistance to other antimalarial drugs in vitro was also examined. Polymerase chain reactions (PCR) for kappa and omega cg2 regions and nested PCR and digestion with ApoI enzyme for K-76T pfcrt point mutation defined corresponding polymorphisms in 83 samples collected between 1995 and 1999. The isotopic microtest was used to evaluate sensitivity in vitro in a subgroup of 18 isolates. The predominant cg2 pattern observed was 13K/14omega repeats (46/83 [55.4%]) and all samples presented the K-76T mutant allele. Seventy-eight percent of samples were resistant to chloroquine in vitro, 35.3% to amodiaquine, 16.7% to mefloquine, and 5.6% to quinine. Significant correlations (P < 0.05) were observed between the IC50s of chloroquine and arteether, and among IC50s of arteether, mefloquine, and quinine. These results suggest the development of multiple and cross-resistance of Colombian P. falciparum isolates to second- and third-line antimalarials and new alternative drugs.     

Chloroquine, sulfadoxine-pyrimethamine and amodiaquine efficacy for the treatment of uncomplicated Plasmodium falciparum malaria in Upper Nile, south Sudan

The current first-line and second-line drugs for Plasmodium falciparum malaria in South Sudan, chloroquine and sulfadoxine-pyrimethamine (SP), were evaluated and compared with amodiaquine, in an MSF-Holland-run clinic in eastern Upper Nile, South Sudan from June to December 2001. Patients with uncomplicated malaria and fever were stratified by age group and randomly allocated to one of 3 treatment regimes. A total of 342 patients was admitted and followed for 14 d after treatment. The dropout rate was 10.2%. Of those who completed the study, 104 were treated with chloroquine (25 mg/kg, 3 d), 102 with SP (25 mg/kg sulfadoxine and 1.25 mg/kg pyrimethamine, single dose) and 101 with amodiaquine (25 mg/kg, 3 d). Adequate clinical response was observed in 88.5% of patients treated with chloroquine, 100% of patients treated with SP and 94.1% of patients treated with amodiaquine. In children aged < 5 years, the success rate was lower: 83.3% for chloroquine and 93.0% for amodiaquine. In adults no treatment failures were found, but children aged 5-15 years showed intermediate levels. In addition, we determined the initial genotypes of dhfr and dhps of 44 isolates from the SP-treated group and > 80% were found to be wild type for dhfr and 100% for dhps. Two percent of isolates had a single mutation and 16% had double mutations of dhfr. These data are in full agreement with the clinical effectiveness of SP. A change in malaria treatment protocols for South Sudan is recommended.     

A randomized comparison of oral chloroquine and sulfadoxine-pyrimethamine for the treatment of uncomplicated Plasmodium falciparum malaria in Laos

Between June and October 2000 we conducted the first randomized trial in Laos comparing chloroquine (CQ) with sulfadoxine-pyrimethamine (SP) in the treatment of uncomplicated Plasmodium falciparum malaria (n = 29, 42-d follow-up, age > 5 years). The proportion of patients with treatment failure was high (CQ = 78%, RIII 46%; SP = 36%, RIII 15%). The treatment policy for uncomplicated P. falciparum malaria in Laos needs to be reviewed urgently.     

Age, temperature, and parasitaemia predict chloroquine treatment failure and anaemia in children with uncomplicated Plasmodium falciparum malaria

The prevalence of chloroquine-resistant Plasmodium falciparum malaria has been increasing in sub-Saharan Africa and parts of South America over the last 2 decades, and has been associated with increased anaemia-associated morbidity and higher mortality rates. Prospectively collected clinical and parasitological data from a multicentre study of 788 children aged 6-59 months with uncomplicated P. falciparum malaria were analysed in order to identify risk factors for chloroquine treatment failure and to assess its impact on anaemia after therapy. The proportion of chloroquine treatment failures (combined early and late treatment failures) was higher in the central-eastern African countries (Tanzania, 53%; Uganda, 80%; Zambia, 57%) and Ecuador (54%) than in Ghana (36%). Using logistic regression, predictors of early treatment failure included younger age, higher baseline temperature, and greater levels of parasitaemia. We conclude that younger age, higher initial temperature, and higher baseline parasitaemia predict early treatment failure and a higher probability of worsening anaemia between admission and days 7 or 14 post-treatment.     

First evidence of pfcrt mutant Plasmodium falciparum in Madagascar

The island of Madagascar, lying in the Indian Ocean approximately 250 miles from the African coast, has so far remained one of the few areas in the world without noticeable Plasmodium falciparum high-grade chloroquine (CQ) resistance. Here we report genotyping data on pfcrt in Madagascar. The pfcrt K76T mutation, which is critical for resistance to CQ, was detected in six (3.3%) of 183 P. falciparum isolates screened, within the mutant haplotypes CVIET and CVIDT. This is the first observation of pfcrt mutant parasites on the island. The current massive distribution of CQ for in-home management of fever in children will promote the dissemination of these mutant CQ-resistant parasites. In this context, genotyping of pfcrt remains a useful tool for CQ resistance surveillance as the prevalence of pfcrt mutations is far from saturation in Madagascar.     

No benefits from combining chloroquine with artesunate for three days for treatment of Plasmodium falciparum in Guinea-Bissau

The use of a combination of chloroquine and artesunate has been suggested for treatment of malaria in Africa. We used concomitant as well as sequential medication with these 2 drugs in relation to each drug separately for children infected with Plasmodium falciparum in Guinea-Bissau from March 2000 to November 2001. By block-randomization, 474 children with symptomatic malaria were divided into 4 groups and given either a total of 8 mg artesunate per kg bodyweight for 3 d, a total of 25 mg chloroquine base per kg bodyweight for 3 d, both drugs concomitantly for 3 d, or both drugs in sequence. All children were followed weekly for 5 weeks. On day 28, parasites had been detected in 40% of the children who were treated with artesunate only compared with 21% treated with chloroquine, 20% treated with artesunate in combination with chloroquine, and 16% treated with artesunate and chloroquine in sequence; on day 35 the corresponding percentages were 48%, 29%, 27%, and 24%, respectively. The outcome of the combination of chloroquine and artesunate in the doses studied was similar to the outcome of chloroquine monotherapy regardless of whether the 2 drugs are given concomitantly (relative risk [RR] = 0.93, 95% CI 0.56-1.53, P = 0.76) or in sequence (RR = 0.78, 95% CI 0.47-1.28, P = 0.32). Thus, neither an antagonistic, an additive, or a synergistic effect of the 2 drugs was indicated.     

Curdlan sulphate in human severe/cerebral Plasmodium falciparum malaria

Preclinical studies have shown that curdlan sulphate (CRDS), a sulphated 1-->3-beta-D glucan, inhibits Plasmodium falciparum in vitro and down-modulates the immune response. A direct, non-specific effect on cytoadherence and rosetting may be predicted, as has been described with other sulphated polysaccharides, e.g. heparin. The anticoagulant effect of CRDS is 10-fold lower than heparin. Curdlan sulphate has, therefore, emerged as a candidate for adjunct medication in the treatment of severe/cerebral malaria. Two clinical studies were conducted using CRDS as adjunct medication to conventional therapy (artesunate) in patients with severe and severe/cerebral malaria. Both studies were double-blind and placebo-controlled to evaluate the efficacy and safety of the combination. Curdlan sulphate appeared to reduce the severity of the disease process, e.g. fever clearance time was shortened. Due to the small number of patients, there was no difference in mortality. The two treatment arms in both studies showed similar results for all laboratory parameters. The only adverse event recorded during CRDS treatment was an increase in activated partial thromboplastin time. This can be monitored easily. It seems that the patients who may benefit most are severe/cerebral cases with no organ damage on admission.     

Complications and mortality patterns due to Plasmodium falciparum malaria in hospitalized adults and children,Rourkela, Orissa,India

Of 1857 Plasmodium falciparum malaria patients hospitalized from 1995 to 1998, 608 had severe malaria and 83 died. Acute renal failure, jaundice and respiratory distress were common in adults whereas children frequently had severe anaemia. Cerebral malaria occurred equally in adults and children but recovery from coma was quicker in children. Multiple complications caused high mortality in adults.     

Artesunate and sulfadoxine-pyrimethamine combinations for the treatment of uncomplicated Plasmodium falciparum malaria in Uganda: a randomized, double-blind, placebo-controlled trial

Drug-resistant malaria is spreading in Africa. The few available drugs might be safeguarded if combined with an artemisinin derivative. We investigated the efficacy, safety, and tolerability of 2 combinations of artesunate with sulfadoxine-pyrimethamine (SP) in a mesoendemic region in Uganda with SP resistance, from September 1999 to June 2000. In a randomized, double-blind, placebo-controlled trial, 420 children aged 6-59 months with uncomplicated Plasmodium falciparum malaria were assigned SP alone (25 mg/kg sulfadoxine, 1.25 mg/kg pyrimethamine) or combined with artesunate (AS; 4 mg/kg/d) for either 1 d (SPAS1) or 3 d (SPAS3). Children were followed-up for 28 d. Day 14 cure rates were 84.6% (99/117) with SPAS3 and 61.9% (73/118) with SPAS1 compared with 55.8% (86/154) with SP. Corresponding day 28 results were 74.4% (87/117) and 45.2% (52/115) compared with 40.5% (62/153). A significant improvement was obtained with the addition of 3 d, but not 1 d, of artesunate (risk ratio [RR] = 1.5, 95% CI 1.3-1.8 at 14 d and RR = 1.8, 95% CI 1.5-2.3 at 28 d). Both AS regimens achieved significantly faster parasite clearance and lower gametocyte carriage. All drug regimens were well tolerated, but SP alone was ineffective. Treatment efficacy improved with SPAS3 but the cure rate at day 28 was modest. The combinations were well tolerated and safe. In areas where SP resistance is prevalent other combinations should be considered.     

Involvement of interleukin-18 in severe Plasmodium falciparum malaria

Serum levels of interleukin-18 (IL-18), interferon-gamma (IFN-gamma), and immunoglobulin E (IgE) were determined for 96 patients with Plasmodium falciparum malaria admitted to hospital, Bangkok, Thailand in the period 1998-2000. The patients were divided into 3 groups, i.e. uncomplicated, severe and cerebral malaria according to WHO criteria (2000). Elevation of IL-18 levels was observed in all 3 groups, with a tendency for higher levels in cases with severe malaria throughout the course of the disease. Moreover, there was a significant correlation between IL-18 levels and the extent of parasitaemia among patients with severe malaria. However, IL-18 levels decreased more significantly in patients with cerebral malaria compared with the other groups in the late stage of the disease. Elevated levels of IFN-gamma were also observed in all groups of patients, especially in those with severe or cerebral malaria, and the levels in patients with cerebral malaria remained significantly higher than in those with uncomplicated malaria during days 4-7 post-treatment, suggesting the involvement of IFN-gamma in disease severity. Meanwhile, no significant difference was observed in IgE levels between the severe and uncomplicated groups of patients with helminth infection, although IgE levels were significantly higher in helminth-infected patients than uninfected patients. These results suggest that IL-18 plays a key role in inducing severe malaria through another pathway, such as elevation of IFN-gamma, rather than its IgE inducing activity.     

Travel as a risk factor for uncomplicated Plasmodium falciparum malaria in the highlands of western Kenya

In the 1980s, highland malaria returned to the tea estates of western Kenya after an absence of nearly a generation. In order to determine the importance of travel for the spread of malaria in this region, we prospectively collected blood films and travel, demographic and geographic information on well persons and outpatients on tea estates near the western rim of the Rift Valley. Risk factors for malaria asexual parasitaemia included: tribal/ethnic group, home province and home district malaria endemicity. Travel away from the Kericho tea estates within the previous two months showed an odds ratio (OR) for parasitaemia of 1.59 for well persons and 2.38 for outpatients. Sexual stages of malaria parasites (gametocytes) had an OR of 3.14 (well persons) and 2.22 (outpatients) for those who had travelled. Increased risk of malaria parasitaemia with travel was concentrated in children aged <5 years. An increase in population gametocytaemia is possibly due to increased chloroquine resistance and suppressed infections contracted outside of the tea estates.     

Changes in susceptibility of Plasmodium falciparum to artesunate in vitro in Yunnan Province, China

We investigated changes in the susceptibility of Plasmodium falciparum to artesunate in vitro using Rieckmann's microtest in Yunnan Province, China, during the period 1988 to 1999. Longitudinal surveillance studies in 1988, 1992 and 1999 revealed that the IC50s were 6.2, 7.2 and 20.7 nmol/L, respectively and mean concentrations completely inhibiting schizont formation (CIMC) were 37.8, 46.1 and 74.0 nmol/L, respectively. The IC50 and CIMC in 1999 were 3.3 and 2 times greater than in 1988. In cross-sectional tests from 1991 to 1993, the susceptibility of P. falciparum isolates from areas in the western and the southern parts of Yunnan Province were similar, but lower than in the south-eastern and central parts of the province. The results suggest that P. falciparum is generally susceptible to artemisinin derivatives but indicate a reduction in susceptibility during the study period in areas where the drugs have been used for a long time.     

Increased frequency of malaria attacks in subjects co-infected by intestinal worms and Plasmodium falciparum malaria

The influence of intestinal worm infections on malaria was studied in individuals from Dielmo, Senegal in 1998. Results suggest that, compared with those infected, individuals free of helminths had the same degree of protection against malaria as that provided by sickle-cell trait, the most potent factor of resistance to malaria identified to date.     

Clinical pattern of severe Plasmodium falciparum malaria in Sudan in an area characterized by seasonal and unstable malaria transmission

A hospital-based study was carried out in Gedarif town, eastern Sudan, an area of markedly unstable malaria transmission. Among the 2488 diagnosed malaria patients, 4.4% fulfilled the WHO criteria for severe malaria, and seven died of cerebral malaria. The predominant complication was severe malarial anemia (45.4%), followed by convulsions (21%), cerebral malaria (16. 4%) and hypotension (11.8%). Severe malaria was recognized in all age groups, but 44.5% of patients were aged 2 to 4 years. The mean ages of patients with severe anemia (5.6 years) and convulsions (5.9 years) were significantly lower than the mean ages of patients with cerebral malaria (14.1 years) or hypotension (35.2 years). Patients with convulsions and cerebral malaria had significantly higher mean parasite count (69972 and 56110 parasites/microL, respectively) than patients with severe anemia (24637 parasites/microL) or hypotension (13667 parasites/microL). The mean blood glucose level was higher in patients with cerebral malaria than in patients with anemia, and higher in patients who died than in patients who survived. In this setting, the clinico-epidemiological pattern of severe malaria varies considerably from that of hyperendemic regions in sub-Saharan Africa, and there is considerable variation between the individual complications of severe malaria.     

Artesunate-atovaquone-proguanil rescue treatment of multidrug-resistant Plasmodium falciparum malaria in pregnancy: a preliminary report

Pregnant women are particularly vulnerable to malaria infections. Multidrug resistance in Plasmodium falciparum seriously compromises treatment in some endemic areas. Between April 1999 and October 2001, we treated and prospectively followed 27 Karen pregnant women with multiple recrudescent P. falciparum infections who were resistant to all other antimalarials with a triple combination of artesunate-atovaquone-proguanil. The treatment was well tolerated and we found no evidence of toxicity for the mothers and the fetus. All but 1 woman were cured (cure rate 96%, 95% CI 89-100). The triple combination of artesunate (4 mg/kg/d), atovaquone (20 mg/kg/d), and proguanil (8 mg/kg/d) may provide a much needed, albeit expensive, 3-d rescue treatment for pregnant women exposed to multidrug- resistant P. falciparum malaria.     

Pharmacodynamic interactions among atovaquone, proguanil and cycloguanil against Plasmodium falciparum in vitro

Synergistic interaction between atovaquone and proguanil has been suggested as the reason for the effectiveness of Malarone. The pharmacodynamic interactions among atovaquone, proguanil and its metabolite cycloguanil were investigated in 4 Plasmodium falciparum parasite strains by culture assays in vitro. The response parameters were determined and 2 statistical methods, log-concentration/response probit method and sum of fractional inhibitory concentrations (sigmaFIC) method, were used to analyse the experimental data. Within therapeutically relevant concentration ratios, the combination of atovaquone and proguanil showed mean sigmaFICs of 0.37 at EC50 (50% effective concentrations) and 0.13 at EC90, indicating high synergism. The combination of atovaquone and cycloguanil yielded corresponding mean sigmaFICs of 3.70 and 2.11, indicating antagonism. The EC50 and EC90 values for proguanil alone were not influenced by RPMI-1640 medium with low concentrations of paraaminobenzoic acid and folic acid (LPLF culture medium), whereas the EC50 and EC90 values for cycloguanil were more than 10 times lower in LPLF medium than in normal RPMI-1640 medium. This confirms the hypothesis that proguanil may act on another target than dihydrofolate reductase. We conclude that the effectiveness of Malarone is due to the synergism between atovaquone and proguanil and may not require the presence of cycloguanil.     

The N-terminal domain of Plasmodium falciparum circumsporozoite protein represents a target of protective immunity

The N-terminal domain of the circumsporozoite protein (CSP) has been largely neglected in the search for a malaria vaccine in spite of being a target of inhibitory antibodies and protective T cell responses in mice. Thus, in order to develop this region as a vaccine candidate to be eventually associated with other candidates and, in particular, with the very advanced C-terminal counterpart, synthetic constructs representing N- and C-terminal regions of Plasmodium falciparum and Plasmodium berghei CSP were administered as single or combined formulations in mice. We show that the antisera generated against the combinations inhibit sporozoite invasion of hepatocytes in vitro better than antisera against single peptides. Furthermore, two different P. falciparum CSP N-terminal constructs (PfCS22-110 and PfCS65-110) were recognized by serum samples from people living in malaria-endemic regions. Importantly, recognition of the short N-terminal peptide (PfCS65-110) by sera from children living in a malaria-endemic region was associated with protection from disease. Taken together, these results underline the potential of using such fragments as malaria vaccine candidates.     

A prospective study of Plasmodium falciparum multiplicity of infection and morbidity in Tanzanian children

Several studies suggest that in individuals with substantial previous exposure to malaria, co-infection with multiple clones of Plasmodium falciparum can protect against subsequent clinical malaria attacks. Other studies, mainly of individuals with little previous exposure, found the converse relationship. To test whether acquisition of such cross-protection tracks the acquisition of clinical immunity in general, 610 Tanzanian children aged 0-6 years were enrolled in a nine-month prospective study of the risk of morbidity in relation to parasitological status and merozoite surface protein 2 genotypes on enrolment. Prevalence of parasitaemia and multiplicity of infection increased with age. In the first year of life, the incidence of clinical malaria was almost three times higher in children with parasites at baseline than in those without. In older children, baseline P. falciparum infections appeared to protect against both parasitaemic and non-parasitaemic fever episodes. In children aged less than three years, baseline multiple infection tended to be associated with higher prospective risk of clinical malaria than single infection while in children aged more than three years the converse was found, but these effects were not statistically significant. These results provide further evidence that relationships between asymptomatic malaria infections and clinical malaria change with cumulative exposure.     

Efficacy of artesunate plus chloroquine for the treatment of uncomplicated malaria in children in Burkina Faso: a double-blind, randomized, controlled trial

Chloroquine (CQ)-resistant Plasmodium falciparum is compromising malaria control in Africa. Combining artesunate (AS) with standard antimalarial drugs increases cure rates and may delay drug resistance. We compared the safety and efficacy of CQ alone and CQ combined with AS (CQ-AS) for treating uncomplicated P. falciparum malaria in Burkina Faso between August 1999 and August 2000. Chloroquine (25 mg/kg over 3 d) combined with AS or placebo (4 mg/kg/d for 3 d) was administered to 300 children aged 6 to 59 months in a randomized, double-blind study. Follow-up extended over 28 d. No adverse drug reactions were recorded. By day 14, parasites were cleared in 120/147 (81.6%) CQ AS-treated children compared with 53/143 (37.1%) CQ-treated children (odds ratio [OR] = 7.55, 95% CI 4.27-13.43, P < 0.001). Corresponding rates for day 28 were 71/145 (49.0%) vs. 27/142 (19.0%) (OR= 4.09, 95% CI 2.33-7.21, P < 0.001). Children who received CQ-AS had significantly faster parasite and fever clearance. Despite the beneficial effects of adding AS, the high failure rate at day 28 of CQ-AS precludes its use as the first-line regimen for treating CQ-resistant P. falciparum in Burkina Faso.